From land to sea: Environmental DNA is correlated with long-term water quality indicators in an urbanized estuary.

Estuaries provide critical ecosystem services, and yet are increasingly under threat from urbanization. Non-invasive approaches to monitor biodiversity resident to or migrating through estuaries is critical to evaluate the holistic health of these ecosystems, often based entirely on water quality. In this study we compared tree of life metabarcoding (ToL-metabarcoding) biodiversity detections with measurements of physico-chemical variables (chlorophyll a, turbidity, total nitrogen, total phosphorous, dissolved oxygen) at eight sites of varying degrees of water quality in the Gold Coast Broadwater Estuary (Queensland, Australia). These sites were ranked according to an adapted Water Quality Index (WQI) score. Here, we detected 787 unique taxa, adding 137 new biodiversity records to the region, mostly micro-organisms such as bacteria, ciliates, diatoms, dinoflagellates, and cryptomonads. Sites with the lowest WQI were characterised by higher turbidity, lower dissolved oxygen, as well as higher total nitrogen and phosphorous, which correlated with an increased diversity of bacteria, ciliates, and green algae. Similarly, the composition of taxa was significantly different between sites with variable WQI values for most taxa but was less apparent for larger vertebrate groups. These findings suggest that rapid ToL-metabarcoding biodiversity detections, particularly for lower order taxonomic groups, can serve as valuable indicators of flora and fauna across the tree of life associated with dynamically shifting estuarine health along urbanized coastlines.

Tree of life metabarcoding can serve as a biotic benchmark for shifting baselines in urbanized estuaries.

Urbanization of estuaries drastically changed existing shorelines and bathymetric contours, in turn modifying habitat for marine foundational species that host critical biodiversity. And yet we lack approaches to characterize a significant fraction of the biota that inhabit these ecosystems on time scales that align with rates of urbanization. Environmental DNA (or eDNA) metabarcoding that combines multiple assays targeting a broad range of taxonomic groups can provide a solution, but we need to determine whether the biological communities it detects ally with different habitats in these changing aquatic environments. In this study, we tested whether tree of life metabarcoding (ToL-metabarcoding) data extracted from filtered seawater samples correlated with four known geomorphic habitat zones across a heavily urbanized estuary (Sydney Harbour, Australia). Using this method, we substantially expanded our knowledge on the composition and spatial distribution of marine biodiversity across the tree of life in Sydney Harbour, particularly for organisms where existing records are sparse. Excluding terrestrial DNA inputs, we identified significant effects of both distance from the mouth of Sydney Harbour and geomorphic zone on biological community structure in the ToL-metabarcoding dataset (entire community), as well as in each of the taxonomic subgroups that we considered (fish, macroinvertebrates, algae and aquatic plants, bacteria). This effect appeared to be driven by taxa as a collective versus a few individual taxa, with each taxon explaining no more than 0.62% of the variation between geomorphic zones. Similarly, taxonomic richness was significantly higher within geomorphic zones with large sample sizes, but also decreased by 1% with each additional kilometer from the estuary mouth, a result consistent with a reduction in tidal inputs and available habitat in upper catchments. Based on these results, we suggest that ToL-metabarcoding can be used to benchmark biological monitoring in other urbanized estuaries globally, and in Sydney Harbour at future time points based on detection of bioindicators across the tree of life. We also suggest that robust biotic snapshots can be archived following extensive curation of taxonomic assignments that incorporates ecological affinities, supported by records from relevant and regional biodiversity repositories.

The spotted parrotfish genome provides insights into the evolution of a coral reef dietary specialist (Teleostei: Labridae: Scarini: Cetoscarus ocellatus).

With over 600 valid species, the wrasses (family Labridae) are among the largest and most successful families of the marine teleosts. They feature prominently on coral reefs where they are known not only for their impressive diversity in colouration and form but also for their functional specialisation and ability to occupy a wide variety of trophic guilds. Among the wrasses, the parrotfishes (tribe Scarini) display some of the most dramatic examples of trophic specialisation. Using abrasion-resistant biomineralized teeth, parrotfishes are able to mechanically extract protein-rich micro-photoautotrophs growing in and among reef carbonate material, a dietary niche that is inaccessible to most other teleost fishes. This ability to exploit an otherwise untapped trophic resource is thought to have played a role in the diversification and evolutionary success of the parrotfishes. In order to better understand the key evolutionary innovations leading to the success of these dietary specialists, we sequenced and analysed the genome of a representative species, the spotted parrotfish (Cetoscarus ocellatus). We find significant expansion, selection and duplications within several detoxification gene families and a novel poly-glutamine expansion in the enamel protein ameloblastin, and we consider their evolutionary implications. Our genome provides a useful resource for comparative genomic studies investigating the evolutionary history of this highly specialised teleostean radiation.

Gut content metabarcoding of specialized feeders is not a replacement for environmental DNA assays of seawater in reef environments.

In tropical marine ecosystems, the coral-based diet of benthic-feeding reef fishes provides a window into the composition and health of coral reefs. In this study, for the first time, we compare multi-assay metabarcoding sequences of environmental DNA (eDNA) isolated from seawater and partially digested gut items from an obligate corallivore butterflyfish (Chaetodon lunulatus) resident to coral reef sites in the South China Sea. We specifically tested the proportional and statistical overlap of the different approaches (seawater vs gut content metabarcoding) in characterizing eukaryotic community composition on coral reefs. Based on 18S and ITS2 sequence data, which differed in their taxonomic sensitivity, we found that gut content detections were only partially representative of the eukaryotic communities detected in the seawater based on low levels of taxonomic overlap (3 to 21%) and significant differences between the sampling approaches. Overall, our results indicate that dietary metabarcoding of specialized feeders can be complimentary to, but is no replacement for, more comprehensive environmental DNA assays of reef environments that might include the processing of different substrates (seawater, sediment, plankton) or traditional observational surveys. These molecular assays, in tandem, might be best suited to highly productive but cryptic oceanic environments (kelp forests, seagrass meadows) that contain an abundance of organisms that are often small, epiphytic, symbiotic, or cryptic.

Gut microbial communities of hybridising pygmy angelfishes reflect species boundaries.

Hybridisation and introgression of eukaryotic genomes can generate new species or subsume existing ones, with direct and indirect consequences for biodiversity. An understudied component of these evolutionary forces is their potentially rapid effect on host gut microbiomes, and whether these pliable microcosms may serve as early biological indicators of speciation. We address this hypothesis in a field study of angelfishes (genus Centropyge), which have one of the highest prevalence of hybridisation within coral reef fish. In our study region of the Eastern Indian Ocean, the parent fish species and their hybrids cohabit and display no differences in their diet, behaviour, and reproduction, often interbreeding in mixed harems. Despite this ecological overlap, we show that microbiomes of the parent species are significantly different from each other in form and function based on total community composition, supporting the division of parents into distinct species, despite the confounding effects of introgression acting to homogenize parent species identity at other molecular markers. The microbiome of hybrid individuals, on the other hand, are not significantly different to each of the parents, instead harbouring an intermediate community composition. These findings suggest that shifts in gut microbiomes may be an early indicator of speciation in hybridising species.

A comprehensive analysis of all known fishes from Sydney Harbour.

Fishes represent an important natural resource and yet their diversity and function in dynamic estuaries with relatively high levels of human pressure such as Sydney Harbour have rarely been quantified. Further, Eastern Australia supports the survival and persistence of an increasing number of tropical species found within temperate estuaries owing to increasing average ocean temperatures. A re-valuation of the number of fish species known from Sydney Harbour is therefore needed. In this study, we generated an up-to-date and annotated checklist of fishes recorded from Sydney Harbour based on verified natural history records as well as newly available citizen science records based on opportunistic observations and structured surveys. We explored the spatial and temporal distribution of these records. In addition, we quantified the function, conservation status, and commercial importance of the identified fishes. The number of fish species recorded from Sydney Harbour now stands at 675, an increase of 89 species (15 %) when compared to the most recent evaluation in 2013. We attribute this increase in fish diversity over a relatively short time to the contribution of newer citizen science programs as well as the influx and survival of fishes in the Harbour with preferences for warmer waters. Some fish families were also overrepresented in the more urbanized and polluted sections of the Harbour. In forecasting further environmental impacts on the fishes of Sydney Harbour, we recommend increased integration of collaborative citizen science programs and natural history collections as a means to track these changes.

Hyporthodus griseofasciatus (Perciformes: Epinephelidae), a new species of deep-water grouper from the west coast of Australia.

A new species of deep-water epinephelid fish is described from the west coast of Australia based on 14 specimens, 99-595 mm standard length. Hyporthodus griseofasciatus sp. nov. is endemic to Western Australia from Barrow Island to Two Peoples Bay in depths of 76-470 m. It has a series of eight grey bands alternating with eight brown bands along the body and the soft dorsal, soft anal and caudal fin margins are pale cream to white. It is distinguished from its nearest congener, H. ergastularius, by the presence of a star-like pattern of radiating lines on the head versus an overall brownish colour in the latter as well as significant differences in the quantitative analyses of 25 morphological characters. The two species have allopatric distributions on either side of the Australian continent. H. griseofasciatus is distinguished from H. octofasciatus by several grey bands being distinctly narrower than other grey bands (vs. all grey bands subequal in the latter) and the presence of broad white margins on the dorsal, caudal and anal fins (vs. narrow or absent in the latter). Some scale counts appear to also differ. Analyses of mitochondrial cytochrome oxidase subunit 1 sequences revealed reciprocally monophyletic clades with fixed differences and genetic distances typical of recently diverged species of fishes.

Demographic histories shape population genomics of the common coral grouper (Plectropomus leopardus).

Many coral reef fishes display remarkable genetic and phenotypic variation across their geographic ranges. Understanding how historical and contemporary processes have shaped these patterns remains a focal question in evolutionary biology since they reveal how diversity is generated and how it may respond to future environmental change. Here, we compare the population genomics and demographic histories of a commercially and ecologically important coral reef fish, the common coral grouper (Plectropomus leopardus [Lacépède 1802]), across two adjoining regions (the Great Barrier Reef; GBR, and the Coral Sea, Australia) spanning approximately 14 degrees of latitude and 9 degrees of longitude. We analysed 4548 single nucleotide polymorphism (SNP) markers across 11 sites and show that genetic connectivity between regions is low, despite their relative proximity (~100 km) and an absence of any obvious geographic barrier. Inferred demographic histories using 10,479 markers suggest that the Coral Sea population was founded by a small number of GBR individuals and that divergence occurred ~190 kya under a model of isolation with asymmetric migration. We detected population expansions in both regions, but estimates of contemporary effective population sizes were approximately 50% smaller in Coral Sea sites, which also had lower genetic diversity. Our results suggest that P. leopardus in the Coral Sea have experienced a long period of isolation that precedes the recent glacial period (~10-120 kya) and may be vulnerable to localized disturbances due to their relative reliance on local larval replenishment. While it is difficult to determine the underlying events that led to the divergence of the Coral Sea and GBR lineages, we show that even geographically proximate populations of a widely dispersed coral reef fish can have vastly different evolutionary histories.

The use of environmental DNA to monitor impacted coastal estuaries.

Environmental DNA (eDNA) metabarcoding is increasingly being used to assess community composition in coastal ecosystems. In this study, we chose to examine temporal and spatial changes in the aquatic community of Manly Lagoon - one of the most heavily developed and polluted estuaries in eastern Australia. Based on metabarcoding of the 16S mitochondrial gene (for fish) and the 18S nuclear gene (for macroinvertebrates), we identified seasonal differences in fish and macroinvertebrate community composition as well as species richness, which correlated, in some cases, with the environmental parameters of sea surface temperature and freshwater input. Moreover, given the greater taxonomic resolution of fish versus macroinvertebrate assignments, we identified several known migratory fish species of management importance that contributed significantly to the overall patterns observed. Overall, our data support the use of eDNA metabarcoding to track fish assemblages shifting in response to environmental drivers in polluted estuaries with increased sampling and consultation with historical data.

Effects of insularity on genetic diversity within and among natural populations.

We conducted a quantitative literature review of genetic diversity (GD) within and among populations in relation to categorical population size and isolation (together referred to as "insularity"). Using populations from within the same studies, we were able to control for between-study variation in methodology, as well as demographic and life histories of focal species. Contrary to typical expectations, insularity had relatively minor effects on GD within and among populations, which points to the more important role of other factors in shaping evolutionary processes. Such effects of insularity were sometimes seen-particularly in study systems where GD was already high overall. That is, insularity influenced GD in a study system when GD was high even in non-insular populations of the same study system-suggesting an important role for the "scope" of influences on GD. These conclusions were more robust for within population GD versus among population GD, although several biases might underlie this difference. Overall, our findings indicate that population-level genetic assumptions need to be tested rather than assumed in nature, particularly for topics underlying current conservation management practices.

Climate-assisted persistence of tropical fish vagrants in temperate marine ecosystems.

Rising temperatures and extreme climate events are propelling tropical species into temperate marine ecosystems, but not all species can persist. Here, we used the heatwave-driven expatriation of tropical Black Rabbitfish (Siganus fuscescens) to the temperate environments of Western Australia to assess the ecological and evolutionary mechanisms that may entail their persistence. Population genomic assays for this rabbitfish indicated little genetic differentiation between tropical residents and vagrants to temperate environments due to high migration rates, which were likely enhanced by the marine heatwave. DNA metabarcoding revealed a diverse diet for this species based on phytoplankton and algae, as well as an ability to feed on regional resources, including kelp. Irrespective of future climate scenarios, these macroalgae-consuming vagrants may self-recruit in temperate environments and further expand their geographic range by the year 2100. This expansion may compromise the health of the kelp forests that form Australia's Great Southern Reef. Overall, our study demonstrates that projected favourable climate conditions, continued large-scale genetic connectivity between populations, and diet versatility are key for tropical range-shifting fish to establish in temperate ecosystems.

Secondary predation constrains DNA-based diet reconstruction in two threatened shark species.

Increasing fishing effort, including bycatch and discard practices, are impacting marine biodiversity, particularly among slow-to-reproduce taxa such as elasmobranchs, and specifically sharks. While some fisheries involving sharks are sustainably managed, collateral mortalities continue, contributing towards > 35% of species being threatened with extinction. To effectively manage shark stocks, life-history information, including resource use and feeding ecologies is pivotal, especially among those species with wide-ranging distributions. Two cosmopolitan sharks bycaught off eastern Australia are the common blacktip shark (Carcharhinus limbatus; globally classified as Near Threatened) and great hammerhead (Sphyrna mokarran; Critically Endangered). We opportunistically sampled the digestive tracts of these two species (and also any whole prey; termed the 'Russian-doll' approach), caught in bather-protection gillnets off northern New South Wales, to investigate the capacity for DNA metabarcoding to simultaneously determine predator and prey regional feeding ecologies. While sample sizes were small, S. mokkaran fed predominantly on stingrays and skates (Myliobatiformes and Rajiformes), but also teleosts, while C. limbatus mostly consumed teleosts. Metabarcoding assays showed extensive intermixing of taxa from the digestive tracts of predators and their whole prey, likely via the predator's stomach chyme, negating the opportunity to distinguish between primary and secondary predation. This Russian-doll effect requires further investigation in DNA metabarcoding studies focussing on dietary preferences and implies that any outcomes will need to be interpreted concomitant with traditional visual approaches.

Phylogenomic Analysis of Concatenated Ultraconserved Elements Reveals the Recent Evolutionary Radiation of the Fairy Wrasses (Teleostei: Labridae: Cirrhilabrus).

The fairy wrasses (genus Cirrhilabrus) are among the most successful of the extant wrasse lineages (Teleostei: Labridae), with their 61 species accounting for nearly 10$\%$ of the family. Although species complexes within the genus have been diagnosed on the basis of coloration patterns and synapomorphies, attempts to resolve evolutionary relationships among these groups using molecular and morphological data have largely been unsuccessful. Here, we use a phylogenomic approach with a data set comprising 991 ultraconserved elements (UCEs) and mitochondrial COI to uncover the evolutionary history and patterns of temporal and spatial diversification of the fairy wrasses. Our analyses of phylogenetic signal suggest that most gene-tree incongruence is caused by estimation error, leading to poor resolution in a summary-coalescent analysis of the data. In contrast, analyses of concatenated sequences are able to resolve the major relationships of Cirrhilabrus. We determine the placements of species that were previously regarded as incertae sedis and find evidence for the nesting of Conniella, an unusual, monotypic genus, within Cirrhilabrus. Our relaxed-clock dating analysis indicates that the major divergences within the genus occurred around the Miocene-Pliocene boundary, followed by extensive cladogenesis of species complexes in the Pliocene-Pleistocene. Biogeographic reconstruction suggests that the fairy wrasses emerged within the Coral Triangle, with episodic fluctuations of sea levels during glacial cycles coinciding with shallow divergence events but providing few opportunities for more widespread dispersal. Our study demonstrates both the resolving power and limitations of UCEs across shallow timescales where there is substantial estimation error in individual gene trees.[Biogeography; concatenation; gene genealogy interrogation; gene trees; molecular dating; summary coalescent; UCEs.].

Does color matter? Molecular and ecological divergence in four sympatric color morphs of a coral reef fish.

Non-sex-linked color polymorphism is common in animals and can be maintained in populations via balancing selection or, when under diversifying selection, can promote divergence. Despite their potential importance in ecological interactions and the evolution of biodiversity, their function and the mechanisms by which these polymorphisms are maintained are still poorly understood. Here, we combine field observations with life history and molecular data to compare four sympatric color morphs of the coral reef fish Paracirrhites forsteri (family Cirrhitidae) in the central Red Sea. Our findings verify that the color morphs are not sex-limited, inhabit the same reefs, and do not show clear signs of avoidance or aggression among them. A barcoding approach based on 1,276 bp of mitochondrial DNA could not differentiate the color morphs. However, when 36,769 SNPs were considered, we found low but significant population structure. Focusing on 1,121 F ST outliers, we recovered distinct population clusters that corresponded to shifts in allele frequencies with each color morph harboring unique alleles. Genetic divergence at these outlier loci is accompanied by differences in growth and marginal variation in microhabitat preference. Together, life history and molecular analysis suggest subtle divergence between the color morphs in this population, the causes for which remain elusive.

Angels in disguise: sympatric hybridization in the marine angelfishes is widespread and occurs between deeply divergent lineages.

Hybridization events are not uncommon in marine environments where physical barriers are attenuated. Studies of coral reef taxa have suggested that hybridization predominantly occurs between parapatric species distributed along biogeographic suture zones. By contrast, little is known about the extent of sympatric hybridization on coral reefs, despite the large amount of biogeographic overlap shared by many coral reef species. Here, we investigate if the propensity for hybridization along suture zones represents a general phenomenon among coral reef fishes, by focusing on the marine angelfishes (family Pomacanthidae). Although hybridization has been reported for this family, it has not been thoroughly surveyed, with more recent hybridization studies focusing instead on closely related species from a population genetics perspective. We provide a comprehensive survey of hybridization among the Pomacanthidae, characterize the upper limits of genetic divergences between hybridizing species and investigate the occurrence of sympatric hybridization within this group. We report the occurrence of hybridization involving 42 species (48% of the family) from all but one genus of the Pomacanthidae. Our results indicate that the marine angelfishes are among the groups of coral reef fishes with the highest incidences of hybridization, not only between sympatric species, but also between deeply divergent lineages.

Marine environmental DNA: Approaches, applications, and opportunities.

Environmental DNA (eDNA) is increasingly being used to document species distributions and habitat use in marine systems, with much of the recent effort focused on leveraging advances in next-generation DNA sequencing to assess and track biodiversity across taxonomic groups. Environmental DNA offers a number of important advantages over traditional survey techniques, including non-invasive sampling, sampling where traditional approaches are impractical or inefficient (e.g. deep oceans), reduced cost, and increased detection sensitivity. However, eDNA applications are currently limited because of an insufficient understanding of the influence of sample source, analytical approach, and marker type on eDNA detections. Because approaches vary considerably among eDNA studies, we present a summary of the current state of the field and emerging best practices. The impact of observed variation in rates of eDNA production, persistence, and transport are also discussed and future research needs are highlighted with the goal of expanding eDNA applications, including the development of statistical models to improve the predictability of eDNA detection and quantification.

Population genomic response to geographic gradients by widespread and endemic fishes of the Arabian Peninsula.

Genetic structure within marine species may be driven by local adaptation to their environment, or alternatively by historical processes, such as geographic isolation. The gulfs and seas bordering the Arabian Peninsula offer an ideal setting to examine connectivity patterns in coral reef fishes with respect to environmental gradients and vicariance. The Red Sea is characterized by a unique marine fauna, historical periods of desiccation and isolation, as well as environmental gradients in salinity, temperature, and primary productivity that vary both by latitude and by season. The adjacent Arabian Sea is characterized by a sharper environmental gradient, ranging from extensive coral cover and warm temperatures in the southwest, to sparse coral cover, cooler temperatures, and seasonal upwelling in the northeast. Reef fish, however, are not confined to these seas, with some Red Sea fishes extending varying distances into the northern Arabian Sea, while their pelagic larvae are presumably capable of much greater dispersal. These species must therefore cope with a diversity of conditions that invoke the possibility of steep clines in natural selection. Here, we test for genetic structure in two widespread reef fish species (a butterflyfish and surgeonfish) and eight range-restricted butterflyfishes across the Red Sea and Arabian Sea using genome-wide single nucleotide polymorphisms. We performed multiple matrix regression with randomization analyses on genetic distances for all species, as well as reconstructed scenarios for population subdivision in the species with signatures of isolation. We found that (a) widespread species displayed more genetic subdivision than regional endemics and (b) this genetic structure was not correlated with contemporary environmental parameters but instead may reflect historical events. We propose that the endemic species may be adapted to a diversity of local conditions, but the widespread species are instead subject to ecological filtering where different combinations of genotypes persist under divergent ecological regimes.

Environmental DNA can act as a biodiversity barometer of anthropogenic pressures in coastal ecosystems.

Loss of biodiversity from lower to upper trophic levels reduces overall productivity and stability of coastal ecosystems in our oceans, but rarely are these changes documented across both time and space. The characterisation of environmental DNA (eDNA) from sediment and seawater using metabarcoding offers a powerful molecular lens to observe marine biota and provides a series of 'snapshots' across a broad spectrum of eukaryotic organisms. Using these next-generation tools and downstream analytical innovations including machine learning sequence assignment algorithms and co-occurrence network analyses, we examined how anthropogenic pressures may have impacted marine biodiversity on subtropical coral reefs in Okinawa, Japan. Based on 18 S ribosomal RNA, but not ITS2 sequence data due to inconsistent amplification for this marker, as well as proxies for anthropogenic disturbance, we show that eukaryotic richness at the family level significantly increases with medium and high levels of disturbance. This change in richness coincides with compositional changes, a decrease in connectedness among taxa, an increase in fragmentation of taxon co-occurrence networks, and a shift in indicator taxa. Taken together, these findings demonstrate the ability of eDNA to act as a barometer of disturbance and provide an exemplar of how biotic networks and coral reefs may be impacted by anthropogenic activities.

Partitioning of diet between species and life history stages of sympatric and cryptic snappers (Lutjanidae) based on DNA metabarcoding.

Lutjanus erythropterus and L. malabaricus are sympatric, sister taxa that are important to fisheries throughout the Indo-Pacific. Their juveniles are morphologically indistinguishable (i.e. cryptic). A DNA metabarcoding dietary study was undertaken to assess the diet composition and partitioning between the juvenile and adult life history stages of these two lutjanids. Major prey taxa were comprised of teleosts and crustaceans for all groups except adult L. erythropterus, which instead consumed soft bodied invertebrates (e.g. tunicates, comb jellies and medusae) as well as teleosts, with crustaceans being notably absent. Diet composition was significantly different among life history stages and species, which may be associated with niche habitat partitioning or differences in mouth morphology within adult life stages. This study provides the first evidence of diet partitioning between cryptic juveniles of overlapping lutjanid species, thus providing new insights into the ecological interactions, habitat associations, and the specialised adaptations required for the coexistence of closely related species. This study has improved our understanding of the differential contributions of the juvenile and adult diets of these sympatric species within food webs. The diet partitioning reported in this study was only revealed by the taxonomic resolution provided by the DNA metabarcoding approach and highlights the potential utility of this method to refine the dietary components of reef fishes more generally.

eDNA metabarcoding survey reveals fine-scale coral reef community variation across a remote, tropical island ecosystem.

Environmental DNA (eDNA) metabarcoding, a technique for retrieving multispecies DNA from environmental samples, can detect a diverse array of marine species from filtered seawater samples. There is a growing potential to integrate eDNA alongside existing monitoring methods in order to establish or improve the assessment of species diversity. Remote island reefs are increasingly vulnerable to climate-related threats and as such there is a pressing need for cost-effective whole-ecosystem surveying to baseline biodiversity, study assemblage changes and ultimately develop sustainable management plans. We investigated the utility of eDNA metabarcoding as a high-resolution, multitrophic biomonitoring tool at the Cocos (Keeling) Islands, Australia (CKI)-a remote tropical coral reef atoll situated within the eastern Indian Ocean. Metabarcoding assays targeting the mitochondrial 16S rRNA and CO1 genes, as well as the 18S rRNA nuclear gene, were applied to 252 surface seawater samples collected from 42 sites within a 140 km2 area. Our assays successfully detected a wide range of bony fish and elasmobranchs (244 taxa), crustaceans (88), molluscs (37) and echinoderms (7). Assemblage composition varied significantly between sites, reflecting habitat partitioning across the island ecosystem and demonstrating the localisation of eDNA signals, despite extensive tidal and oceanic movements. In addition, we document putative new occurrence records for 46 taxa and compare the efficiency of our eDNA approach to visual survey techniques at CKI. Our study demonstrates the utility of a multimarker metabarcoding approach in capturing multitrophic biodiversity across an entire coral reef atoll and sets an important baseline for ongoing monitoring and management.